[MUSIC] My name is Mogens Vyberg. I'm Professor of clinical pathology, and have been working with liver pathology for more than 40 years. Liver biopsy remains the criterion standard for the diagnosis of non-alcoholic fatty liver disease. Here we see the needle biopsy material. Ideally it should have a length of at least 20 millimeters, and a thickness of approximately 1 millimeter, in order to get a sufficient number of portal tracts. In this video, I'll show you what we look for, when we examine liver biopsies in the diagnosis of non-alcoholic fatty liver disease. This disease starts with steatosis, which in some cases develops to non-alcoholic steatohepatitis, that may show progressive fibrosis, ending up in cirrhosis. In the ladder, hepatocellular carcinoma may develop. The photo to the left shows a normal dark brown liver. To the right is seen a steatotic yellowish enlarged liver. The end stage of progressive fibrosis namely cirrhosis, is as shown here nodular, the nodular consist of liver cells surrounded by fibrous bands. To the right the hepatocellular carcinoma is found. Fatty liver disease including steatosis and steatohepatitis may have several courses. The best known is alcohol. The histopathologic entity, non-alcoholic steatohepatitis, as a metabolic disease is relatively new. Few decades ago, the morphological liver changes similar to what we now know as non-alcoholic steatohepatitis, were primarily identified in patients with large alcohol consumptions. And the term alcoholic hepatitis, often coin to these changes, sometimes even though the patient has little or no known alcohol intake. It should however be emphasized that rarely drug-induced liver lesions, for example due to amiodarone and inherent metabolic diseases like Wilson disease, may reveal similar changes. Here you see a case of severe steatosis. Liver steatosis is an accumulation of triglycerides. In histologic sections of formalin-fixed paraffin-embedded liver biopsy, this is seen as backyards, that are distinct, in other words sharply deiodinaded and empty. Backhoes are small or large holes in the liver of cellsido plasmin, that are created by extraction of the fat during the tissue preparation. The term macrovesicular steatosis, used when the droplets are large, are usually a metabolic feature. The term microvesicular steatosis, indicating that the droplets are mainly small, may in contrast be caused by various drugs and toxins. When grading macrovesicular steatosis, an estimated proportion of liver cells with fat vacuoules less than 5%, is not included. Slight steatosis is defined as 5-32% of the liver cells involved, moderate as 33-66% and severe as atleast 67% of the liver cells involved. Steatosis is normally considered to be harmless lesion. However, it may be the starting point of non-alcoholic steatohepatitis, which is potentially a progressive disease, hovering the risk of developing to cirrhosis. The differential diagnosis between steatosis and non-alcoholic steatohepatitis is therefore important, and can only be made by histological examination. In non-alcoholic fatty liver disease, steatosis is macrovascular or mixed. Some of the large stellates do not contain fat, but are ballooned as shown here. Ballooning is hydropic degeneration of liver cells, which typically become rounded in the (inaudible) with a diffusely clear cytoplasma as shown here. Ballooning is due to oxidative cell stress. It starts in the centrilobular areas and is one of the features required to classify the liver lesion as non-alcoholic steatohepatitis. The ballooned cells sometimes contain scattered (inaudible) uniformly condensed also called hyaline components in the cytoplasm. The data may be gathered to form so called mallory-denk bodies, which are aggregates of cytoplasmic filaments, primarily carotenes type 8 and 18. Also inflammation or inflammatory infiltration in the liver is a required feature of NASH. In this field we see diffuse infiltration of lymphocytes and maybe some other cells like histiocytes and neutrophils. The inflammation is followed by fibrosis lack here. It may be difficult actually to see fibrosis to identify it in hematoxylin eosin stains. In the picrosirius red staining to the left, fibrosis is easily found. The collagen fibers produced by the perisinusoid hepatic stellate cells, also called the eater cells, are first located in the perisinusoidal space but during the progression, the fibers are spread in a pericellular, the so-called chicken-wire pattern. As the damaged liver cells die and disappear, the coding strands merge, creating scepter that speed up the development. Mellory-denk bodies as well as their precursors can mainly be seen in precursors staining, but nicely visualized in immunohistochemical, stands for the regulatory protein, ubiquitin, which targets proteins to the proteasome that degrades and recycles proteins. These images are from a study we performed almost 30 years ago, where we disdained the hematoxylin eosin stain slides and restrained for ubiquitin to show how much easier it was to identify the mellory-denk bodies. Here in the liver with severe steatohepatitis the four stains, hematoxylin eosin, sirius red ubiquitin and the ubiquitin-binding sequestosome 1, also called ubiquitin-binding p62 protein aligned illustrating the ballooning pericellular fibrosis and mallory-denk bodies. And here is a larger magnification of another case of severe steatohepatitis Here we find heavy infiltration around the cells containing mallory-denk bodies, which can be identified in all four stains. This image shows fully developed cirrhosis with (inaudible) nodules surrounded by fibrous bands. In the lower part of the photo a preserved central vein is shown embedded in fibrous tissue. Hepatocellular carcinoma as shown here is a severe complication of NASH with cirrhosis. In biopsies the identification of hepatocellular carcinoma may be challenging. In some cases, the tumor cells are highly differentiated, resembling non-neoplastic cells. However, they usually grow in irregular thickened trabeculae. To the left the normal tissue, to the right the neoplastic tissue. Here are two slides from a normal liver and hepatocellular carcinoma respectively has been silver stained for reticulin. In the normal liver to the left, a normal to regular pattern is seen demarked by complete reticulin strands. While in the other irregular trabeculae are demarked with incomplete reticulin strands. In the (inaudible) cells are normally CD34 positive, but mostly in the (inaudible) cells covering hepatic sinusoids are as an excipient, CD34 negative. To the left you can see normal liver with the CD34 positive in the (inaudible) cells covering large vessels and the sinusoids close to the portal tract, while there is no CD34 reaction in the rest of the liver sinusoids. In contrast in hepatocellular carcinoma sinosoids are spaces, in fact transformed to cabularies are outlined within the (inaudible) cells expressing CD34. In other cases, hepatocellular carcinoma resembles metastatic carcinomas. Identification of the hepatocellular origin as here shown, may be aided by the immunohistochemicall staining pattern. Most important positive (inaudible) markers are arginase 1, an enzyme functioning in the urea cycle, and glypican 3, an oncofetal proteoglycan. Formation of bile canaliculi is a diagnostic feature of most hepatocellular carcinomas. Bile canaliculi may be visualized by, for example CD10, which is a certain membrane peptidase. The staining pattern within branching structures is shown to the left, in some cases mallory-denk bodies as well, are developed in the tumor cells and can be identified or visualized with immunostains for ubiquitin or P62. As you have seen in this presentation, liver biopsy is a valuable tool to identify and classify non-alcoholic fatty liver disease. To assess the progression of the disease and to identify complications, like hepatocellular carcinoma. The distinction between steatosis and steatohepatitis is important. It must be based on histology, even though the method is not perfect. [MUSIC]
